Progression of DCIS to invasive breast cancer through CCR2 chemokine signaling

描述（由申请人提供）：导管原位癌（DCIS）是女性中最常见的非浸润性乳腺癌类型，是浸润性导管癌（IDC）的直接前兆。目前的细胞和组织病理学方法不能准确预测疾病进展，导致患者对DCIS治疗不足或过度治疗。我们的长期目标是确定导致IDC的关键因素，从而能够开发基于分子的方法来预测IDC的风险，以及更有针对性的治疗DCIS的方法。DCIS中的CCL 2/CCR 2趋化因子受体信号传导是DCIS进展为IDC的潜在重要机制。CCR 2及其结合配体CCL 2在乳腺导管癌中过表达，并与肿瘤分级和患者预后不良相关。CCL 2/CCR 2趋化因子信号传导最为人所知的是调节巨噬细胞募集。然而，CCL 2的敲低显著抑制乳腺肿瘤进展而不显著影响巨噬细胞募集。在导管癌细胞中CCR 2的过表达增强细胞存活和侵袭性，与促存活和促侵袭蛋白（ALDH 1A 1）的表达增加和促凋亡蛋白（Htra 2）的表达减少相关。这些研究表明CCR 2信号在乳腺导管癌进展中的重要作用。为了阐明CCL 2/CCR 2信号传导的体内作用，我们通过人乳腺癌细胞系Sum 225和DCIS.com的小鼠乳腺导管内注射（MIND）开发了新型异种移植模型。Sum 225 MIND模型形成稳定的非侵入性病变，而DCIS病变在DCIS.com MIND模型中进展为IDC。与传统的移植模型相比，这些MIND模型更接近于模拟患者中发现的DCIS生物学。与Sum 225病变相比，DCIS.com病变显示出更高的CCL 2、CCR 2和ALDH 1A 1蛋白表达以及更低的Htra 2表达，这与向浸润性癌的进展相关。基于初步研究，我们假设导管癌细胞中的CCL 2/CCR 2信号传导促进DCIS向IDC的进展。目标1：使用原代和已建立的乳腺癌细胞系，通过分析MIND模型、3D细胞培养模型和生化方法中CCR 2过表达和敲低的影响，表征DCIS期间导管癌细胞中自分泌CCL 2/CCR 2信号传导的作用。目标二：通过使用肾下移植物移植模型、MIND模型、3D细胞培养模型和生物化学方法，使用原代和已建立的乳腺癌细胞系，分析来源于成纤维细胞的CCL 2对DCIS进展的影响，确定基质来源的CCL 2对CCR 2介导的DCIS进展的功能贡献。通过多PI计划，目的是确定CCL 2/CCR 2信号传导如何调节促侵袭和促存活因子（ALDH 1A 1）和促凋亡因子（Htra 2）的表达，以促进导管癌进展。这些研究将揭示趋化因子受体信号传导在DCIS中的作用机制，具有重要的治疗意义。.

DESCRIPTION (provided by applicant): Ductal carcinoma in situ (DCIS) is the most common type of non-invasive breast cancer diagnosed in women and an immediate precursor to invasive ductal carcinoma (IDC). Current cyto- and histopathological approaches do not accurately predict disease progression, resulting in patients being under-treated or over-treated for DCIS. Our long-term goals are to identify key factors that lead to IDC that will enable the development of a molecular based approach to predict the risk of IDC, and a more tailored approach to treat DCIS. CCL2/CCR2 chemokine receptor signaling in DCIS is a potentially important mechanism for DCIS progression to IDC. CCR2 and its binding ligand CCL2, are overexpressed in breast ductal carcinomas, and correlate with tumor grade and poor patient prognosis. CCL2/CCR2 chemokine signaling is best known for regulating macrophage recruitment. However, knockdown of CCL2 significantly inhibits mammary tumor progression without significantly affecting macrophage recruitment. Overexpression of CCR2 in ductal carcinoma cells enhances cell survival and invasiveness, associated with increased expression of pro-survival and pro-invasive proteins (ALDH1A1) and decreased expression of pro-apoptotic proteins (Htra2). These studies indicate an important role for CCR2 signaling in breast ductal carcinoma progression. To clarify the in vivo role of CCL2/CCR2 signaling, we have developed novel xenograft models through mouse Mammary intraductal injection (MIND) of human breast carcinoma cell lines, Sum225 and DCIS.com. The Sum225 MIND model form stable non-invasive lesions, while DCIS lesions progress to IDC in the DCIS.com MIND model. Compared to conventional transplant models, these MIND models more closely mimic the biology of DCIS found in patients. Compared to Sum225 lesions, DCIS.com lesions show higher expression of CCL2, CCR2 and ALDH1A1 proteins and lower expression of Htra2, correlating with progression to invasive carcinoma. Based on preliminary studies, we hypothesize that CCL2/CCR2 signaling in ductal carcinoma cells enhances progression of DCIS to IDC. Aim 1: To characterize the role of autocrine CCL2/CCR2 signaling in ductal carcinoma cells during DCIS by analyzing effects of CCR2 overexpression and knockdown in MIND models, 3D cell culture models and biochemical approaches, using primary and established breast cancer cell lines. Aim 2: To determine the functional contribution of stromal derived CCL2 on CCR2 mediated progression of DCIS, by analyzing the effects of CCL2 derived from fibroblasts on DCIS progression, using subrenal graft transplant models, MIND models, 3D cell culture models and biochemical approaches, using primary and established breast cancer cell lines. Through a multiple PI plan, the objective is to determine how CCL2/CCR2 signaling regulates expression of pro-invasive and pro-survival factors (ALDH1A1) and pro-apoptotic factors (Htra2) to promote ductal carcinoma progression. These studies will reveal mechanistic insight into the role of chemokine receptor signaling in DCIS, with important therapeutic implications. .